Načrtovanje in sinteza C-6 substituiranih derivatov glukozamina kot zaviralcev MurA encima

Brilej, Neža (author), Frlan, Rok (mentor)

Odpornost na antibiotike je eden večjih svetovnih zdravstvenih izzivov, zato je vse večja potreba po razvoju novih protibakterijskih učinkovin ter odkritju novih tarč. Kot odlična tarča se je že v preteklosti izkazal encim MurA, ki sodeluje v zgodnji fazi sinteze peptidoglikana bakterij. Vloga MurA pri biosintezi peptidoglikana je prenos enolpiruvata s fosfoenolpiruvata na UDP-N-acetilglukozamin. MurA je ključen za preživetje bakterijske celice, ni pa prisoten v celicah sesalcev, zato je iz vidika selektivnosti idealna protibakterijska tarča. Kot edini registrirani zaviralec MurA je na voljo fosfomicin, vendar so bakterije nanj odporne v vse večji meri. V magistrski nalogi smo s posnemanjem osnovnega substrata UDP-N-acetilglukozamina poskusili doseči zaviralni učinek na encimu MurA. Kot osnovni skelet smo izbrali N-acetilglukozamin, na katerem smo izvajali kisle modifikacije na mestu C-6, ki bi potencialno tvorile reverzibilne interakcije s cisteinom v aktivnem mestu encima. V sklopu raziskovalnega dela smo se osredotočali na razvoj sintezne poti reverzibilnih zaviralcev MurA ter na koncu sintetizirali devet končnih spojin, od katerih smo na petih izvedli biološko testiranje zaviralne aktivnosti na Escherichia coli, vendar nobena od njih ni izkazala zadostnega zaviranja na encimu MurA. Izhajali smo iz N-acetil glukozamina. Sintezo smo pričeli z zaščito prostih hidroksilnih skupin, na mestu C-1 smo jo zaščitili z metiliranjem, medtem ko smo na mestih C-3 in C-4 uporabili benzilno zaščito. OH skupino na mestu C-6 smo na začetku zaščitili s tritilno zaščito, ki smo jo po zaključenem benziliranju odstranili s kislo hidrolizo, saj smo na tem mestu pripravljali modifikacije. V naslednji stopnji smo izvedli oksidacijo s TEMPO in BAIB do karboksilnega derivata, kjer smo dalje s sklopitvenimi reagenti tvorili amidno vez z aminokislinama (glicin in β-alanin). Do derivata s prosto amino skupino smo prišli preko aktivacije OH skupine s tozil kloridom, ki smo ga v naslednji stopnji pretvorili v azid, katerega smo reducirali do NH2. Na tem mestu smo kasneje tvorili derivate s sukcinsko ter oksalno kislino.

• Language: Slovenian
• Keywords: bakterijska odpornost, MurA, peptidoglikan, N-acetilglukozamin
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-158582
• Publication date in RUL: 16.06.2024

Secondary language

• Language: English
• Title: Design and synthesis of C-6 substituted glucosamine derivatives as MurA enzyme inhibitors

Abstract

Antibiotic resistance is one of the major global health challenges, demanding development of new antibacterial agents and discovery of new targets. The enzyme MurA, which participates in the early stage of bacterial peptidoglycan synthesis, has previously proven to be an excellent target. MurA's role in peptidoglycan biosynthesis involves transferring enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine. The enzyme is essential for bacterial cell survival but is absent in mammalian cells, making it an ideal antibacterial target in terms of selectivity. Fosfomycin is the only registered MurA inhibitor available at the moment, but bacteria are increasingly becoming resistant to it. In our master's thesis, we attempted to achieve inhibitory effects on the MurA enzyme by mimicking its basic substrate UDP-N-acetylglucosamine. We chose N-acetylglucosamine as the base and performed acidic modifications on the C-6 position, which could potentially form reversible interactions with cysteine in the enzyme's active site. As part of the research, we focused on development of a synthrtic pathway of reversible MurA inhibitors and managed to synthesize a total of nine final compounds, of which we conducted biological testing for inhibitory activity against E. coli on five. However, none of them exhibited sufficient inhibition. We started from N-acetylglucosamin. Initially we protected free hydroxyl groups, on C-1 position by methylation and using benzyl protecting groups for on the C-3 and C-4 positions. The hxdroxyl group on C-6 spot was initially protected with tritiyl protection, which was removed by acidic hydrolisis after benzylation, as this position was planned for further modifications. In the next step, TEMPO and BAIB oxidation was preformed to achieve carboxylic derivate, which was followed by the formation of amide bond with amino acids (glycine and β-alanine) using coupling agents. Free amino group derivate was achieved by activating the hydroxyl group with tosyl chloride, which was converted to azide and reduced to NH2. At this stage derivates with succinic and oxalic acid were formed.

• Keywords: bacterial resistance, MurA, peptidoglycan, N-acetylglucosamine